Immune-mediated allergic (hypersensitivity) reactions are classified into four types (I-IV) according to the underlying mechanisms leading to the expression of the allergic symptoms. Type I allergic reactions are characterized by IgE-mediated release of vasoactive substances such as histamine from mast cells and basophils. The release of these substances and the subsequent manifestation of allergic symptoms are initiated by the cross-linking of allergen-bound IgE to its receptor on the surface of mast cells and basophils.
An IgE antibody is a complex molecule consisting of two identical heavy chains and two identical light chains held together by disulfide bonds in a “Y” shape-configuration. Each light chain consists of a variable (VL) domain linked to a constant domain (CL), and each heavy chain consists of a variable domain (Vh) and four constant domains (CH1, CH2, CH3, and CH4, also known as Cε1, Cε2, Cε3, and Cε4; respectively). The two arms of an IgE antibody contain the site at which an IgE antibody binds to its specific antigen (allergen) and each arm is referred to as a Fab (fragment-antigen-binding) fragment. The tail of an IgE antibody is termed Fc (fragment-crystalline) as it can form crystals when separated from the Fab fragments of the antibody under appropriate experimental conditions. The Fc fragment of an IgE antibody consists of the Cε2, Cε3, and Cε4 domains and contains the biologically active structures of the IgE antibody (e.g., receptor binding sites).
The production of IgE antibodies requires interactions and collaborations among three cells; antigen presenting cells (APC), T lymphocytes (T helper cells; Th) and antibody producing cells (B lymphocytes; B cells). When a foreign substance, an allergen, is introduced for the first time into the body of subjects (e.g., by inhalation of environmental allergen, ingestion of certain foods, or via the skin), the allergen is taken up by APCs (e.g., macrophages) which then digest or process the allergen into smaller fragments (epitopes). These fragments are displayed on the surface of APCs in association with specific molecules known as major histocompatibility complex proteins (MhC). The allergen fragment/MhC complex displayed on the surface of APCs is recognized and bound by receptors on the surface of specific T lymphocytes. This recognition and binding event leads to the activation of T lymphocytes and the subsequent expression and secretion of cytokines such as interleukin-4 (IL-4). These cytokines induce the multiplication, clonal expansion and differentiation of B cells specific for the allergen in question (i.e., B cell which express on their surface immunoglobulin receptors capable of binding to the allergen) and ultimately lead to the production of IgE antibodies from these B cells. A portion of the activated T lymphocytes and IgE producing B cells eventually become committed to a pool of cells called T and B memory cells, which are capable of faster recognition of allergen upon subsequent exposure to the allergen.
In individuals suffering from type I allergic reactions, exposure to an allergen for a second time leads to the production of high levels of IgE antibodies specific for the allergen as a result of the involvement of memory B and T cells in the 3-cell interaction required for IgE production. The high levels of IgE antibodies produced during the second exposure lead to cross-linking of IgE receptors on mast cells and basophils by allergen-bound IgE, which in turn leads to the activation of these cells and the release of the pharmacological mediators that are responsible for the clinical manifestations of type I allergic diseases.
Two receptors with differing affinities for IgE have been identified and characterized. The high affinity receptor (FcεRI) is expressed on the surface of mast cells and basophils. The low affinity receptor (FcεRII/CD23) is expressed on many cell types including B cells, T cells, macrophages, eosinophils and Langerhan cells. The high affinity IgE receptor consists of three subunits (alpha, beta and gamma chains). Several studies demonstrate that only the alpha chain is involved in the binding of IgE, whereas the beta and gamma chains (which are either transmembrane or cytoplasmic proteins) are required for signal transduction events. The identification of IgE structures required for IgE to bind to the FcεRI on mast cells and basophils is of utmost importance in devising strategies for treatment or prevention of IgE-mediated allergies. For example, the elucidation of the IgE receptor-binding site could lead to the identification of peptides or small molecules that block the binding of IgE to receptor-bearing cells in vivo.
Over the last 15 years, a variety of approaches have been utilized to determine the FcεRI binding site on IgE. These approaches can be classified into five different categories. In one approach, small peptides corresponding to portions of the Fc part of an IgE molecule were produced and analyzed for their ability to inhibit IgE from its receptors. See, for example, Nakamura et al., EP0263655 published Apr. 13, 1988, Burt et al., 1987, European Journal of Immunology, 17:437-440; helm et al., 1988, Nature 331:180-183; helm et al., 1989, PNAS 86:9465-9469; Vercelli et al., 1989, Nature 338:649-651; Nio et al, 1990, Peptide Chemistry, 2: 203-208; Nio et al., 1993, FEBS Lett. 319:225-228; and Nio et al., 1992, FEBS Lett. 314:229-231. Although many of the peptides described in these studies were shown to inhibit the binding of IgE to its receptors, different studies reported different sequences as being responsible for IgE binding.
helm et al. (1988, Nature 331:180-183) identified a 75 amino acid peptide that spans the junction between C,h2 and C,h3 domains of IgE and showed that this peptide binds to the IgE receptor with an affinity close to that of the native IgE molecule. On the other hand, Basu et al. (1993, Journal of Biological Chemistry 268: 13118-13127) expressed various fragments from IgE molecules and found that only those fragments containing both the CH3 and CH4 domains were able to bind IgE and that CH2 domain is not necessary for binding. Vangelista et al. (1999, Journal of Clinical Investigation 103:1571-1578) expressed only the CH3 domain of IgE and showed that this domain alone could bind to IgE receptor and prevent binding of IgE to its receptor. The results of Basu et al. and Vangelista et al. are inconsistent and conflict with those of helm et al. cited above.
In a second approach to identify the FcRI binding site on IgE, polyclonal antibodies against peptides corresponding to parts of the CH2 domain, CH3 domain or CH4 domain were produced and used to probe for receptor binding site on IgE (Robertson et al., 1988, Molecular Immunol. 25:103-118). Robertson et al. concluded that the amino acid residues defined by a peptide derived from the CH4 domain were not likely to be involved in receptor binding, whereas amino acid residues defined by a peptide derived from the CH3 domain of IgE were most likely proximal to the IgE receptor-binding site (amino acids 387-401) however, the anti-CH3 peptide antibodies released histamine from IgE-loaded mast cells indicating that the amino acids defined by the CH3 peptide did not define the bona fide IgE receptor-binding site and that anti-CH3 peptide antibodies could cause anaphylaxis.
In a third approach to identify the FcεRI binding site on IgE, several investigators produced IgE mutants in an attempt to identify the amino acid residues involved in receptor binding (see, e.g., Schwarzbaum et al., 1989, European Journal of Immunology 19:1015-1023; Weetall et al., 1990, Journal of Immunology 145:3849-3854; and Presta et al., 1994, Journal of Biological Chemistry 269:26368-26373). Schwartzbaum et al. demonstrated that a mouse IgE antibody with the point mutation proline to histidine at amino acid residue 442 in the CH4 domain has a two fold reduced affinity for the IgE receptor. Schwartzbaum et al. concluded that the CH4 domain of an IgE antibody is involved in IgE binding to its receptor, however, Schwartzbaum's conclusion contradict Weetall et al.'s conclusion that the binding of mouse IgE to its high affinity receptor involves portions of the CH2 and CH3 domains of the IgE antibody, but not the CH4 domain. Further, Schwartzbaum et al.'s conclusions contradict Presta et al.'s conclusion that the amino acid residues of the IgE antibody important for binding to the FcεRI are located in the CH3 domain.
In a fourth approach to identify the FcεRI binding site on IgE, chimeric IgE molecules were constructed and analyzed for their ability to bind to the FcεRI. Weetall et al., supra constructed a series of chimeric murine IgE-human IgG molecules and tested their binding to the IgE receptor. Weetall et al., supra concluded that the CH4 domain does not participate in receptor binding and that the CH2 and CH3 domains are both required for binding to the high affinity receptor on mast cells. In another study, Nissim et al. (1993, Journal of Immunol 150:1365-1374) tested the ability of a series of human IgE-murine IgE chimera to bind to the FcεRI and concluded that only the CH3 domain is needed for binding to the FcεRI. The conclusion by Nissim et al. corroborates the conclusion by Vangelista et al. that the CH3 domain of IgE alone binds to the FcεRI. However, the conclusions by Nissim et al. and Vangelista et al. contradict the conclusions of Weetall et al. and Robertson et al.
Presta et al., supra produced chimeric human IgG in which the CH2 was replaced with CH3 from human IgE. When tested for receptor binding, this chimera bound to the FcεRI albeit with a four-fold reduced affinity compared with native IgE. The results of Presta et al. appear to corroborate with the results of Nissim et al., but conflict with those of Weetall et al., helm et al., and Basu et. al., cited above. In a further attempt to define the exact amino acid residues responsible for the binding of IgE to its receptor, Presta et al. inserted specific amino acid residues corresponding to CH2-CH3 hinge region and three loops from the CH3 domain of human IgE into their analogous locations within human IgG and called these mutants IgGEL. Unfortunately, when these IgGEL variants were tested for receptor binding, they exhibited minimal binding compared to the native IgE or the IgG in which the full length CH3 domain replaced the full length CH2 domain.
In a fifth approach to identify the FcεRI binding site on IgE, monoclonal antibodies have been developed and analyzed for their ability to block IgE binding to the FcεRI. See, for example, Del Prado et al., 1991, Molecular Immunology 28:839-844; Keegan et al., 1991, Molecular Immunology 28:1149-1154; hook et al., 1991, Molecular Immunology 28:631-639; Takemoto et al., 1994, Microbiology and Immunology 38:63-71; and Baniyash et al., 1988, Molecular Immunology 25:705-711. Although many monoclonal antibodies have been developed, they have provided little information on the bona fide IgE receptor-binding site because in many cases the amino acid sequence recognized by these monoclonal antibodies have not or could not be identified. Further, the monoclonal antibodies developed may block IgE from binding to its receptor by steric hindrance or induction of severe conformational changes in the IgE molecule, rather than by the binding and masking of IgE residues directly involved in receptor binding.
It is apparent from the above discussion that approaches that have been devised to identify the receptor binding site on IgE have produced conflicting results. The difficulty in the identification of the amino acid residues of IgE responsible for receptor binding could be further complicated by the possibility that the site on IgE used for binding to the receptor may not be a linear sequence of amino acids, which could be mimicked by a synthetic peptide. Rather, the binding site may be a conformational determinant formed by multiple amino acids that are far apart in the IgE protein sequence which are brought into close proximity only in the native three-dimensional structure of IgE. Studies with IgE variants, IgE chimera, and monoclonal anti-IgE antibodies strongly suggest that the binding site is a conformational determinant.
Currently, IgE-mediated allergic reactions are treated with drugs such as antihistamines and corticosteroids which attempt to alleviate the symptoms associated with allergic reactions by counteracting the effects of the vasoactive substances released from mast cells and basophils. high doses of antihistamines and corticosteroids have serious side effects such as renal and gastrointestinal toxicities. Thus, other methods for treating type I allergic reactions are needed.
One approach to the treatment of type I allergic disorders has been the production of monoclonal antibodies which react with soluble (free) IgE in serum, block IgE from binding to its receptor on mast cells and basophils, and do not bind to receptor-bound IgE (i.e., they are non-anaphylactogenic). Two such monoclonal antibodies (rhuMab E25 and CGP56901) are in advanced stages of clinical development for treatment of IgE-mediated allergic reactions (see, e.g., Chang, T. W., 2000, Nature Biotechnology 18:157-62). The identity of the amino acid residues of the IgE molecule recognized by these monoclonal antibodies are not known and it is presumed that these monoclonal antibodies recognize conformational determinants on IgE.
Although early results from clinical trials with therapeutic anti-IgE monoclonal antibodies suggest that these therapies arc effective in the treatment of atopic allergies, the use of monoclonal antibodies for long-term treatment of allergies has some significant shortcomings. First, since these monoclonal antibodies were originally produced in mice, they had to be reengineered so as to replace mouse sequences with consensus human IgG sequences (Presta et al., 1993, The Journal of Immunology 151:2623-2632). Although this “humanization” process has led to production of monoclonal antibodies that contain 95% human sequences, there remain some sequences of mouse origin. Since therapy with these anti-IgE antibodies requires frequent administration of the antibodies over a long period of time, some treated allergic patients could produce an antibody response against the mouse sequences that still remain within these therapeutic antibodies. The induction of antibodies against the therapeutic anti-IgE would negate the therapeutic impact of these anti-IgE antibodies at least in some patients. Second, the cost of treatment with these antibodies will be very high since high doses of these monoclonal antibodies are required to induce a therapeutic effect. Moreover, the frequency and administration routes with which these antibodies have to be administered is inconvenient. A more attractive strategy for the treatment of IgE-mediated disorders is the administration of peptides which induce the production of anti-IgE antibodies.
One of the most promising treatments for IgE-mediated allergic reactions is the active immunization against appropriate non-anaphylactogenic epitopes on endogenous IgE. Stanworth et al. (U.S. Pat. No. 5,601,821) described a strategy involving the use of a peptide derived from the CH4 domain of the human IgE coupled to a heterologous carrier protein as an allergy vaccine. However, this peptide has been shown not to induce the production of antibodies that react with native soluble IgE. Further, hellman (U.S. Pat. No. 5,653,980) proposed anti-IgE vaccine compositions based on fusion of full length CH2-CH3 domains (approximately 220 amino acid long) to a foreign carrier protein. however, the antibodies induced by the anti-IgE vaccine compositions proposed by hellman will most likely result in anaphylaxis since antibodies against some portions of the CH2 and CH3 domains of the IgE molecule have been shown to cross-link the IgE receptor on the surface of mast cell and basophils and lead to production of mediators of anaphylaxis (see, e.g., Stadler et al., 1993, Int. Arch. Allergy and Immunology 102:121-126). Therefore, a need remains for vaccines for the treatment of IgE-mediated allergic reactions, which do not induce anaphylactic antibodies.
The significant concern over induction of anaphylaxis has resulted in the development of another approach to the treatment of type I allergic disorders consisting of mimotopes that could induce the production of anti-IgE polyclonal antibodies when administered to animals (see, e.g., Rudolf, et al., 1998, Journal of Immunology 160:3315-3321). Kricek et al. (International Publication No. WO 97/31948) screened phage-displayed peptide libraries with the monoclonal antibody BSWI7 to identify peptide mimotopes that could mimic the conformation of the IgE receptor-binding site. These mimotopes could presumably be used to induce polyclonal antibodies that react with free native IgE, but not with receptor-bound IgE as well as block IgE from binding to its receptor. Kricek et al. disclosed peptide mimotopes that are not homologous to any part of the IgE molecule and are thus different from peptides disclosed in the present invention.
A major obstacle facing the development of an anti-IgE vaccine is the lack of information regarding the precise amino acids representing non-anaphylactogenic IgE determinants that could be safely used to immunize allergic subjects and induce non-anaphylactogenic polyclonal antibodies (i.e., polyclonal anti-IgE antibodies that do not bind to receptor-bound IgE). The peptide compositions of the present invention are selected to be non-anaphylactogenic; i.e., the peptide compositions do not result in production of anti-IgE antibodies that could cause cross-linking of IgE bound to mast cells or basophils. Thus peptides of the present invention have superior safety profile and are differentiated by sequence composition from disclosed vaccines based on full-length C2h-CH3 domains.
The safety and efficacy of therapies intended for treatment of IgE-mediated allergies are usually evaluated in animal models such as mice, rats and dogs. A variety of mouse and rat models have been developed for several types of IgE-mediated allergies such as asthma, atopic dermatitis and food allergies (Xin-Min Li, et al.; J. Allergy Clin. Immunol 1999; 103:206-214, Xui-Min et al.; J. Allergy Clin Immunol., 2001, 107:693-702). Although these models have been useful in evaluation of small molecule-based treatment modalities, they are not suitable for evaluation of vaccine-based treatments. This is because the IgE-derived peptide epitope(s) that are used for development of a vaccine for non-rodent species e.g. dogs, can be significantly different from those of mice and rats. Although naturally occurring canine models of allergies are available (e.g. Ermel R W, et al.; Laboratory Animal Science 1997, 47:40-48), these models take a very long time to develop and only a limited number of animals are available at one time. Furthermore, once these dogs are used for a vaccine trial, they cannot be used for further trials. Although dogs can be experimentally sensitized to allergens such as flea allergens (e.g. McDermott, M J, et al.; Molecular Immunology, 2000; 37:361-375), the limitations discussed above still apply. Thus, an appropriate method to induce high levels of IgE and clinical signs of Type I hypersensitivity in dogs is needed to allow rapid evaluation of vaccines and other therapies for treatment of allergies in the desired target species.
Ricin is a lectin found in castor beans which has been found to enhance IgE production directed against a variety of antigens. For example, administration of ricin in conjunction with an antigen can boost the production of IgE in rats that are inherently low in IgE (e.g. Underwood, S L et al.; Immunology. 1995;85:256-61, Underwood, S L et al.; Int Arch Allergy Immunol. 1995;107:119-21 and Diaz-Sanchez D. et. al.; Immunology. 1991;72:297-303). Several studies have determined that ricin enhances IgE responses by preferentially inhibiting a population of activated CD8+ T lymphocytes. These CD8+ cells are thought to express counter regulatory cytokines (e.g. interferon gamma) that down regulate the Th2 cytokines (IL-4, IL-10, and IL-5) released by CD4+ lymphocytes that provide class-switching signals for B-lymphocytes to express IgE (Noble A, et al.; Immunology 1993, 80:326 and Diaz-Sanchez, D. et. Al.; Immunology. 1993;78:226-236.). Previous studies also show that IgE responses to bee venom phospholipase A2 were reduced by 90% in rats receiving an adoptive transfer of the immunosuppressive CD8+ T lymphocytes (Diaz-Sanchez et al.; Immunology 1993, 78:226-236). Compared to CD4+ cells, this population of regulatory CD8+ T lymphocytes has high affinity receptors for the ricin lectin. Following entry of the lectin into the activated cell, cellular protein synthesis is inhibited resulting in killing of the cell. Rats immunized with antigen and ricin show a dramatic increase in the CD4/CD8 ratio due to a 40% decrease in CD8+ T lymphocytes occurring between 7 and 21 days after immunization ((Diaz-Sanchez et al.; Immunology 1993, 78:226-236).
Thus to facilitate and accelerate the development of allergy models, there is a need as provided in the method of the present invention, for induction of high levels of IgE and concomitant induction of clinical signs of allergies in normal dogs following simultaneous exposure to allergens and ricin. This method utilizes normal dogs, which are readily available, and results in sensitization of the majority of dogs in a relatively short period of time.